During the current reporting period we have: (1) coauthored a paper that recently appeared online in Arthritis and Rheumatology describing the NIH experience on the use of the recombinant IL-1 receptor antagonist, anakinra, in the treatment of CAPS (the cryopyrin-associated periodic syndromes) in pregnancy; (2) submitted a paper describing the role of the NLRP3 inflammasome in APLAID (autoinflammation and PLC-gamma2-associated antibody deficiency and immune dysregulation); (3) submitted a paper describing a myeloid-specific NLRP3 somatic mutation in a patient with adult-onset CAPS; (4) utilized whole-exome sequencing to screen for de novo dominant mutations underlying undiagnosed autoinflammatory conditions; (5) collaborated with NIAMS investigators in the description of a new autoinflammatory condition, denoted SAVI (STING-associated vasculopathy with onset in infancy), caused by de novo dominant mutations in TMEM173, encoding the stimulator of interferon genes (STING). This work was recently published in the New England Journal of Medicine. Anakinra in Pregnancy A total of 9 women (4 with FCAS, 1 with MWS, and 4 with NOMID) reported 1 to 4 pregnancies each, resulting in a total of 15 FCAS, 3 MWS, and 6 NOMID pregnancies. Six births from FCAS mothers and 3 births from NOMID mothers occurred while patients were receiving anakinra. If a woman became pregnant while taking anakinra, the pre-pregnancy anakinra dose was continued. The anakinra dose was increased during 1 twin pregnancy. No preterm births or serious complications of pregnancy were observed. One fetus of the twin pregnancy had renal agenesis and suffered fetal demise. Genetic testing showed the deceased twin carried the same NLRP3 p.Val262Ala mutation as the mother. The other twin is healthy and mutation-negative. From this experience we concluded that anakinra provided significant, persistent CAPS symptom relief while continuing to prevent the long-term sequelae of CAPS; that anakinra was well-tolerated; and that although a causal relationship between anakinra and renal agenesis seems unlikely, further safety data are needed. The NLRP3 Inflammasome in APLAID In the previous reporting period we demonstrated that a novel variant, p.Ser707Tyr, in phospholipase Cgamma2 (PLCg2), is the cause of APLAID. The hypermorphic mutation enhances the PLCg2 activity and causes an increase in intracellular calcium release from endoplasmic reticulum stores. In the previous reporting period we also showed that increased intracellular calcium signaling causes NLRP3 inflammasome activation. We therefore examined the role of the NLRP3 inflammasome in the pathogenesis of APLAID. We isolated human peripheral blood mononuclear cells (PBMCs) from two affected patients and 3 healthy controls. We analyzed inflammasome activation by Western blotting, and measured intracellular calcium levels with the FLIPR Calcium 4 assay kit. We found that patients cells had elevated basal levels of intracellular calcium and that intracellular calcium flux triggered by extracellular calcium chloride was substantially enhanced. Patients PBMCs secreted IL-1beta in response to LPS priming alone, and this effect was attenuated by the use of a PLC inhibitor, intracellular calcium blockers, or an adenylate cyclase activator. Our findings suggest that the inflammation in patients with APLAID is partially driven by the activation of the NLRP3 inflammasome. The data link two seemingly distinct molecular pathways and provide new insights into the pathogenesis of APLAID and autoinflammation. A manuscript describing these findings was submitted to Arthritis and Rheumatology, and is accepted pending minor revisions. NLRP3 Somatic Mutation in Adult-Onset CAPS A 52 year-old pediatrician presented to our clinic with the recent onset, about at the time of menopause, of stress-induced fevers, chills, urticaria, fatigue, and profound myalgia, usually lasting several hours at a time. We performed whole-exome sequencing and targeted deep resequencing using DNA from the patients whole blood to identify a possible NLRP3 somatic mutation. We then screened for this mutation in subcloned NLRP3 amplicons from fibroblasts, buccal cells, granulocytes, and negatively-selected monocytes and T and B lymphocytes. We identified a previously reported CAPS-associated mutation, p.Tyr570Cys, with a mutant allele ratio of 15% based on exome data. Targeted resequencing of NLRP3 confirmed the presence of the somatic mutation in whole blood, but not in buccal cells, at a ratio similar to the exome data. The mutated allele was present in 26.6% and 30.4% of monocytes and granulocytes, respectively, while this mutation was not present in B lymphocytes, T lymphocytes, buccal cells, or in the patients cultured fibroblasts. The data document the possibility of myeloid-restricted somatic mosaicism in the pathogenesis of CAPS, underscoring the emerging role of massively parallel sequencing in clinical diagnosis. A manuscript describing these findings has been submitted to Arthritis and Rheumatology. Whole-Exome Sequencing in Undiagnosed Autoinflammatory Conditions We are currently utilizing whole-exome sequencing in cases of undiagnosed autoinflammatory disease. We select for cases with a relatively early age of onset, and with relatively dramatic phenotypes. Whole-exome sequencing of DNAs from patients and their unaffected parents (and at times other family members) is performed at the NIH Intramural Sequencing Center (NISC), and data are analyzed either under the assumption of recessive inheritance or de novo dominant mutations. In one set of 29 such cases, we have identified 15 plausible candidate genes. In followup, we are performing targeted sequencing of these candidate genes in a panel of 100 other unexplained patients from our clinic, on the hypothesis that we may find additional mutations in the 15 candidate genes in other patients with similar phenotypes. In selected cases we are also performing functional studies to further test the possible link between variant and disease. Preliminary data suggest a possible connection between variants of TNFAIP3, a gene involved both in NF-kappaB activation and apoptosis, and susceptibility to a Behcets disease-like phenotype. During the upcoming reporting period we will screen additional patients with this phenotype for TNFAIP3 variants, we will perform functional studies on patients cells and transfected cell lines, and we will attempt to study the effects of such variants in animal model systems. STING-Associated Vasculopathy with Onset in Infancy Our contribution to this project involved patient samples, but all of the laboratory studies were conducted or coordinated by our colleague, Dr. Raphaela Goldbach-Mansky, and we will therefore leave the description of this project to her Annual Report. A manuscript describing these findings was recently published in the New England Journal of Medicine.